Process of forming adherent films on animal tissue

ABSTRACT

Animal tissue, such as bruised skin, skin graft removal sites, corneas, and the like are coated with a protective layer of undenatured collagen by electrophoretic migration of collagen particles from a suspension thereof in contact with the tissue. The layer thus formed is adherent, and in general becomes a part of the animal treated.

United States Patent Inventor Maurice Seiderman 3306 Derondo Drive,Hollywood, Calif. 90028 Appl. No. 803,421

Filed Feb. 28, I969 Patented Feb. 16, 1971 PROCESS OF FORMING ADHERENTFILMS ON ANIMAL TISSUE 6 Claims, No Drawings US. Cl L/ l 3/l,128/334;2Q4/18l;264/24, 264/222 Int. Cl A6lb 19/00 Field ofSearch 128/334,

305.5, 335.5, 335; 3/l;204/180, 181, (inquired); l 17/(inquired);1815.1; 264/(lnquired), 24, 222

[56] References Cited UNITED STATES PATENTS 3,443,261 5/1969 Battista etal. 128/1X 3,454,966 7/1969 Rosen 3/13 3,491,760 l/l 970 Braun et al.128/334 OTHER REFERENCES Adduct Detection Electrophoresis" G. Reich,Collagen Currents p. 249 Vol. 5, No. 6, Dec, 1964. Copy in Group 330Primary Examiner- Dalton L. Truluck Assistant Examiner-J. YaskoA!t0rneyHerzig & Walsh '1 PROCESS OF FORMING ADIIEREN'I FILMS N ANIMALTISSUE This invention relates to coating animal tissue with collagensomewhat after the manner of a skingraft, but in a simplified andimproved fashion utilizing suspensions of undenatured collagen. f

The need frequently arises for coating animal tissue with a skinlikelayer which in favorable cases becomes solidly attached on to the tissuein question. Skin grafts are of course well known, but to avoidrejection phenomena are generally taken from the same animal, and inaddition are not employable in the case of certain specialized tissues,such as the cornea, where it is naturally essential that the graftbecome and remain transparent. j

An object of the present invention is to provide a method of making askin or tissue graft, whereby a layer of collagen is implaced on thetissue to be treated in such a fashion that it is adherent, dense, andskinlike.

Other objects of the invention'will appear as the discussion thereofproceeds.

Generally speaking, and in accordance with illustrative embodiments ofmy invention, 1 commence with a suspension of comminuted undenaturedcollagen, as more particularly described herein below; and I bring thissuspension into contact with the tissue to be treated. Where the treatedarea is very small, surface forces between the tissue and the electrodeto be described are generally sufiicient to maintain the suspension inplace; and where larger areas are treated, it isa simple matterto form adam surrounding the tissue to be treated, so that the suspension remainsin place by gravity; or where the tissue to be treated forms part of ananimal extremity,thelater may simply be immersed in a container togetherwith the suspension.

. I then place a first electrode intoelectricalcontact with thesuspension, and preferably reasonably closeto the tissue to be treated.Also, I place a second electrode inelectricalcontact with the animal, asby strapping it to any convenient part such as a hand or leg, for whichthe routine techniques of electrocardiography may be used. Them! impressa voltage on the two electrodes, the first electrode being positive andthe second electrode being negative. The suspended collagen particlesare then acted upon by .two essentially electrical forces: because theyare themselves dipoles, they tend to become aligned with the lines offorce of the electrical fluid; and second, because they possess anoverall positive charge, they commence to migrate toward-the treatedtissue. When they reach the treated tissue, they are unable to migratefurther, and a layer of collagen is built up which moreover is onehaving the collagen particles or fibrils aligned, so that a dense andadherent skinlike layer is formed, and attached in intimate contact withthe tissue. y

The electrodeposition as described is carried out until a layer ofdesired thickness has been formed, whereupon the apparatus is removedfrom the animal treatedLln general no sub sequent treatment of the layeris needed, except to allow it to The preparation of thesuspension ofdenatured collagen is known to the art. For example, bovine deep flexortendon may .be cleaned and trimmed of fat and other extraneous matter,then frozen, and sliced perpendicular to the longitudinal axis of thetendons, a suitable thickness being from 0.2 mm. 0.5 mun, although thisis not critical. The collagen fibrils may then be disaggregated bytreatment with a proteolytic enzyme such as commercial ficin, forexample by-gentle agitation for an hour at 35 C. in an aqueous solutionof ficin. They are then washed with distilled water, and treated with 1percent neutral aqueous sodium chloride solution for one hour in twosuccessive treatments, added to a mixture of equal parts of methanol andwater containing 2 percent cyanoscetic acid, agitated at near freezing,and then homogenized by a conventional homogenizer. However; manyalternative procedures are known, such as that using aqueous carbonicacid solutions and fully described in U.S. Pat. No. 3,368,911

A suitablecollagen is that comminuted by the process of Battista (J.Appl. Polymer Sci. l.l 48l-498 (l967) and described in the articlecited. The individual fibrils of this product are as short as onemicron, or even somewhat shorter.

The suspension of comminuted undenatured collagen may be kept for sometime before use, preferably in a refrigerator at close to freezingtemperature. The 'electrodeposition, however, is carried out preferablyat room temperature. Indeed, some local heating of the solution takesplace as a result of the passage of the electrical current.

It will be apparent to those skilled in the art that the voltage andcurrent requirements are quite dependent upon the dimensional parametersin any given treatment, such as the area to be coated with the adherentfilm of collagen, the area of the electrodes, the separation of theelectrode from the animal tissue, and the mean temperature. It isdesirable to pro vide a voltage supply variable over the range of about1 to about 50 volts. The actual current requirements are of coursesmall, generally measurable as a few millamperes. In general it is bestto work at lower rather than higher voltages, which favorselectrophoresis over electrolysis, some of the latter being inevitablealthough reducible to a minimum by avoiding the presence of electrolytesin the collagen suspension as far as possible. Also, working atrelatively low voltages, and thus taking a relatively longer time suchas a few minutes instead of a few secondsyto deposit the collagen filmavoids an increase in'temperature which, if carried too high, maycommence to denature'the collagen.

Collagen from many sources is suitable, such as mammalian collagen fromsheep and horses. However, I prefer bovine collagen, because of itsready availability and quite general acceptability,-that isnonrejection, by the animal host.

The comminution of the collagen refers, of course, to reduction in thelength of the fibrils by the primary cutting process, since when placedinto suspension the individual, elementary fibrils are separated fromeach another by the known techniques already described. Otherwisestated, the diameter of the fibrils is dependent only upon the source.As stated, a .practical upper limit for their length of the comminutedfibril is0.5 to 0.6 mm. The fibrils may of course be much shorter thanthis, limited only by the capability of the comminuting device orprocess used, and as mentioned, may be as short as a fraction of amicron.

The concentration of the collagen in the suspension is likewise variablewithin wide limits. Below about one-tenth percent by weight collagen inwater leads to impracticably long deposition times, with repeatedreplenishment of the suspension being required. Suspensions moreconcentrated than 2 or 3 percent tend to become so viscous thatmigration of the particles is impeded. l have found suspensions of from0.25 percent to 1 percent by weight best.

The thickness of the layer deposited on the animal tissue inaccordancewith the invention may be varied within wide limits. Athickness of less than 0.1 mm. is scarcely enough to achieve anypractical result. Most frequently, thicknesses of about 0.2 to 0.5 mm.will be found suitable for most purposes.

An illustrative working example follows. The subject is a rabbit havinga portion of the skin about 2 cm? in area abraded from the foreleg. Aone-half percent suspension of beef tendon collagen, sliced at 0.4 mm.fibril length is prepared using the ficin method described above. Theentire foreleg is immersed in a tray containing the suspension, acontact electrode is placed on a hind foot, the electrode being densegraphite and using a 5 percent potassium chloride agar gel to obtainelectrical conductivity. A dense graphite electrode, having a face 3 cm.in diameter, is maintained about 1 cm. from the abraded portion,immersed in the collagen suspension. A current is passed in the manneralready described, using a voltage of about 5 volts, for 5 minutes, atthe end of which time an adherent collagen film has been built up on theabraded portion. The foreleg; is then rinsed free of unused collagen anddried in air.

A particular advantage of the process in accordance with the inventionis that when an abraded portion of dermal tissue is treated, forexample, the collagen fibrils deposited at the periphery of the abradedarea tend to interlock with the rough edges of the intact collagenalready present. Thus,

not only does the deposited collagen layer adhere to the central portionof the tissue treated as already described, but there comes into play avery favorable mechanical effect of peripheral interlocking, whichassists subsequent cicatrization.

While I have described my invention with the aid of various specificexamples, it will be understood that I do not desire to be limited tothe exact details described for obvious modifications will occur topersons skilled in the art.

lclaim:

l. The process of coating animal tissue with a layer of collagen whichcomprises the steps of:

placing a suspension of comminuted undenatured collagen in contact withsaid tissue;

emplacing a first electrode in electrical contact with said suspension;

emplacing a second electrode in electrical contact with said animal; and

impressing a voltage between said electrodes, said first electrode beingpositive and said second electrode being negative, and maintaining saidvoltage until a preselected thickness of said layer is formed on saidtissue.

2. The process in accordance with claim 1 wherein said collagen ismammalian.

3. The process in accordance with claim 1 wherein said collagen is beeftendon collagen.

4. The process in accordance with claim 1 wherein said collagen ispresent in the form of fibrils having a length of not to exceed about0.6 mm. v

5. The process in accordance with claim 1 wherein said tissue is dermaltissue.

6. The process in accordance with claim 1 wherein said tissue is cornealtissue.

2. The process in accordance with claim 1 wherein said collagen ismammalian.
 3. The process in accordance with claim 1 wherein saidcollagen is beef tendon collagen.
 4. The process in accordance withclaim 1 wherein said collagen is present in the form of fibrils having alength of not to exceed about 0.6 mm.
 5. The process in accordance withclaim 1 wherein said tissue is dermal tissue.
 6. The process inaccordance with claim 1 wherein said tissue is corneal tissue.